To search for lens-derived neuroprotection of RGCs, these neurons
were identified by retrograde tracing. To do this, 48 rats received the
fluorescent dye D291,
N-4-[4-didecylaminostryryl]-
N-methyl-pyridinium
iodide (4Di-10ASP; Molecular Probes, Eugene, OR) into the SC.
Figure 1 shows the anatomic relationships within the eye and the projection of
ganglion cell axons through the ON to the SC. Eight days later, the ON
contralateral to the injected SC was surgically exposed in its
intraorbital aspect and mechanically crushed at 0.5 mm beyond the ON
head for 10 seconds, as described earlier. The rats were then divided
into six groups. The first group (
n = 6) received ON crush,
intraocular penetration and injury of the lens (perforation routes 1
and 2,
Fig. 1 ) containing the lens proliferative zone of the
epithelium, and injection of 10 μl phosphate-buffered saline (PBS, pH
7.4) according to standard protocols.
13 The second group
(
n = 6) received ON crush, retrolenticular intraocular
penetration (perforation route 3,
Fig. 1 ), and injection of 10 μl PBS
without lens damage. A third group of control animals (
n = 6) received ON-crush, but no eye injection or lens injury. A fourth
group (
n = 18) received ON-crush, with lens injury plus PBS
(perforation route 2,
Fig. 1 ) by 3, 5, or 7 days (six animals per
subgroup) after ON-crush. The fifth group of control rats (
n= 6) received only retrograde labeling of RGCs but no further
manipulation at the ON or in the eye. This group was used to determine
the numbers of retrogradely filled RGCs, and the rats were killed 8
days after the staining procedure. A group of six rats underwent
labeling only and were killed at 22 days after staining—that is, after
the total study period of the other experimental groups—to assure that
the long-term staining did not affect cell survival by itself. Finally,
a group of five rats were treated the same as the animals of the fourth
group, but without PBS injection to test the effect of the injection
route alone.